Method for preparing reticulo-endo-thelial system stimulant and stimulant thereof



United States Patent 0 METHGD FQR FREEARRNG RETIQULQJEND S- THELIAL SYSTEM dTiMULANT AND STIMU- LANT THEREGE Alfred E. Fen, Haclrettstown, Benjamin S. Schwartz, Livingston, and Frank Turner, Succasunna, Ni, assignors to Warner-Lambert Pharmaceutical orn= parry, Morris Plains, Nil a corporation of Delaware No Drawing. Filed Apr. 24, 13 62, Ser. No. 189,696

2 Claims. (Cl. 167-78) This invention relates to a process for separating and fractionating cell wall of mycobacteria into useful components, and relates more particularly to a new and novel process for the separation, extraction and purification of the cell wall of mycobacterium, such as Mycobacterium plzlei, into components, and pa ticularly into a component fraction which is highly effective in stimulating the activity of the reticulo-endothelial system of a host to whom this active fraction is administered.

It is widely recognized that many noticeable di ferences exist in the susceptibility of individuals towards the adverse or toxic effects commonly produced by invading microorganisms. These differences are believed to refiect variations in the available body defense mechanisms, one of the most important of which is the reticulo-endothelial system. The reticulo-endothelial system comprises, for example, reticulo-endothelial cells of the spleen pulp,

ells of the lymphatic system, certain liver cells and reticulo-endothelium cells of the bone marrow.

The reticulo-endothelial system acts in the following ways to protect the host from invading microorganisms: (1) by phagocytosis; (2) by digestion of the phagocytized bacteria; (3) by the production of specific antibodies; and (4) by the production of non-specific defense factors, such as properdin and complement.

It is, therefore, an important object of this invention to isolate an agent which will stimulate the reticulo-endothelial system and thus non-specifically enhance the natural defense mechcanisms of the host. The non-specific action of this agent may be contrasted to the introduction of a specific antigen, such as a polio vaccine. In this case, only one function, the production of a specific antibody, is the result of this type of stimulation. it is our purpose to accomplish a general stimulation of all of the functions of the reticulo-endothelial system.

The phagocytic activity of the reticulo-endothelial system is a factor which can be measured experimentally. Thus, for example, by introducing a controlled amount of colloidal carbon particles, of a specific and uniform size, into the blood stream of test animals and measuring the time necessary for their removal by phagocytosis, a standard clearance curve may be determined. This curve is obtained by plotting the log of the carbon concentration, in the sample removed, against time and the slope of the resulting line is denoted as the carbon clearance rate, or K value. it is apparent that when phagocytosis is stimulated, the K value increases accordingly. Although the carbon clearance method provides a means for determining phagocytic stimulation, it does not measre the degree of protection afforded the host against invading microorganisms as a result of this stimulation. This parameter may be evaluated, for example, by the intravenous administration, to selected groups of mice, of a lethal dose, for controls, of Salmonella enterizidz's. The administration of the Salmonella enteritldis is made to one group of mice at a preselected number of days following the completion of treatment with a stimulant for the reticulo-endothelial system and parallel administration is made to a control group of untreated mice. The percent mortalities which result are plotted versus the time, in days, following the injection of the Salmonella enteritirlis. The time, in days, at which fifty percent of the untreated group died is compared with the time, in days, at which fifty percent of the treated group died. The value determined in this manner is designated as the median survival time (MST The significance of MST and its relation to the present invention will become more apparent after the following detailed description.

While different materials will stimulate the reticuloendothelial system, the essential criteria are that they must be non-toxic, easily metabolized and non-allergenic, since otherwise serious side effects could be produced. The value of stimulating the phagocytic activity of the reticulo-endothelial system and increasing the speed of motion or chemotaxis of these cells in their attack on foreign matter is obvious. This stimulation offers a new mechanism for speedily utilizing the natural defense mechanisms of the body.

it is, furthermore, an important object of this invention to provide a non-toxic, easily metabolized and non-allergenic agent capable of stimulating the reticulo-endothelial system and which can be used for enhancing the natural defense mechanisms of the host.

Another object of this invention is to provide a process for preparing this agent involving the rupture, separation, isolation and subsequent purification of the cell wall components of mycobacterium.

Other objects of this invention will appear from the following detailed description.

It has been found that when live mycobacteria are injected intravenously into mice, they produce a marked increase in the resistance of that host to subsequent experimental infection.

For example, it has been observed that in the case of Swiss mice of 20 to 25 gm. body weight, which have re ceived Mycobacterium phlel, there results a seven-fold increase in the carbon clearance rate over untreated mice. In the protection test against Salmonella enteritldis infection, for example, those mice which have received Mycobacterium phlei prior to infection are found to have a six day increase in median survival time. It is apparent, therefore, that living mycobacteria, as in the case of Mycobacterium plzlei, stimulate the reticulo-endothelial system and are potentially valuable agents for enhancing the natural host defense mechanisms.

The use of whole, live organisms to produce a reticuloendothelial stimulation does entail several drawbacks. Among these are the allergenicity and toxicity of the intact, viable cells. It is, therefore, quite apparent that it would be advantageous to isolate the particular active component, or components, from mycobacteria responsible for this stimulation, free of allergenic and toxic properties.

We have found that certain cell-wall components of mycobacteria contain a specific reticulo-endothelial stimulating factor and that in accordance with the novel process of this invention these cell-wall components may be isolated and purified, thus making available a versatile reticulo-endothelial system stimulant.

To obtain the desired cell wall components, for example, the cells must first be broken or ruptured in order to release the intracellular components, and the latter must be separated from the cell wall components before they can be fractionated to separate the active material.

It will be readily appreciated by those skilled in the art that bacterial cells can be ruptured by physical or chemical means. Thus, ultrasonic waves may be employed in some instances to break up bacteria to achieve sterilization. The use of hypertonic saline will also cause some cells to lyse. Other methods, such as the passage of cells through a small orifice under high pressure, have also been described. While these methods of rupturing cells are effective in achieving their purposes, they are not desirable when the cell wall components of mycobacteria are the desired end product. Furthermore, there has not been, as yet, any eiiective isolation process which can be employed economically and efliciently in separating cell wall and cell wall components of mycobacteria from the cytoplasmic components of ruptured cells. As can be readily appreciated, ordinary means of separation, such as filtration, cannot be employed satisfactorily in order to separate the components of a colloidal suspension, such as that containing both cell wall and cytoplasmic components. Ex traction methods using solvents have been demonstrated to be undesirable due to alteration of some of the basic properties of this material.

The novel process of this invention enables complete rupturing of the cells of mycobacteria and eiiective isolation of the desired cell wall components. This can be done, for example, by subjecting a suspension of 6.5 to 7.0 percent of dry mycobacteria, in water, to attrition by passage through a particle size reducer such as a colloid mill. The force of attrition on the cell wall is so great under these conditions that they become totally deformed and rupturing of the cell is effected. To achieve as complete a rupturing of all of the cells as possible, for example, superfine glass beads of an average dimension of 120 microns are added to the cell suspension. This applies a greater impact on the cells while they are being sheared. To avoid the disadvantages of cell clumping, a surface active agent such as Tween 80 is preferably added as a 0.5% solution in water. Thus, a suspension of mycobacteria in water containing the glass beads and surface active agent may be totally ruptured by continuous passage through a colloid mill for about 25 minutes. The suspension of the cell wall components and cytoplasmic material thus obtained is then collected in a flask. The total rupture of the cells by this treatment is readily demonstrated by the use or application of stains to the treated mixture and microscopic examination.

Since the known methods of separating a colloidal bacterial suspension into the several components are not effective in this case, a novel isolation method has been devised whereby just the components of the cell wall which are active in stimulating the reticulo-endothelial system are obtained. Thus, by subjecting the ruptured cell sus pension to controlled gravitational forces, we have found that phase separation occurs at 9,200 to 11,000 times gravity and that the sediments thus obtained show maximum protection in mice against Salmonella enteritidis infection. Since the suspension is already separated into a sediment and a supernate portion by the controlled gravitational forces applied and the active material is in the sediment, the inactive material which remains in the supernatant fluid is simply decanted. In order to separate any whole cells that may not have been ruptured, the sediment, which is obtained in the initial separation, is resus pended in water. The reconstituted suspension is subjected to a controlled gravitational force of approximately 1,000 to 1,200 times gravity whereby any remaining intact cells will sediment and the supernatant fluid, which contains the desired cell wall components, is decanted. This enables one to obtain, as a suspension, the crude, active reticuloendothelial system stimulant. The total solids present in the crude suspension are then determined by placing a measured aliquot in a tared vessel and then drying it at 90 C. until a constant weight is obtained.

The crude suspension may also contain undesirable materials, such as nucleic acid and various proteins. To achieve homogeneity and purity, the cell wall components are then preferably digested with an enzyme such as ribonuclease, which destroys the nucleic acid, and then with a second enzyme to remove protein. Trypsin may be employed as the protein digestant.

Based upon the total solids present in the crude suspension, one part, by Weight, of ribonuclease is added to twenty parts, by weight, of total solids present in that suspension. The resulting suspension is then incubated at 37 C. for 60 minutes. At the conclusion of incubation, it is subjected to a gravitational force of 9,200 to 11,000 times gravity for 30 minutes at a temperature of 5 C. the sediment while the enzyme remains in solution in the supernatant fluid and is simply decanted. In order to remove any trace of enzyme, the sediment is washed three times by resuspending in water and subjected to a gravitational force of 9,200 to 1l,000 gravity. The same procedure as described above is repeated using 2.5 parts, by weight, of trypsin to 8 parts, by weight, of total solids present after initial digestion with ribonuclease. The total solids present after ribonuclease and trypsin digestion are again determined by the method described above. The material is resuspended in water or physiological saline to contain 2.0 mg./rnl. and is injected intravenously into mice at a dose of 10 to 20 mg./kg. of body weight.

In order to further illustrate this invention, but Without being limited thereto, the following examples are given:

Example I To 100 ml. of sterile Dubos broth containing 10 ml. of Dubos medium albumin, is added aseptically a loopful of Mycobacterium phlei, maintained as a stock culture on Lowenstein agar. The resulting suspension is then incubated on a rotary shaker at 37 C. for 40 to 48 hours. At the end of this incubation, 1.0 ml. of the bacterial suspension is withdrawn aseptically into a fresh, sterile 100 ml. of Dubos broth, containing 10 ml. of Dubos medium albumin. The resulting suspension is incubated at 37 C. for 72 to 76 hours on a rotary shaker. At the end of this incubation period, 0.5 ml. of the bacterial suspension is transferred into a Roux bottle containing 200 ml. of Sautons medium. The inoculated Roux bottle is then incubated stationary at 37 C. for 10 days. At the end of 10 days, the bacterial suspension is harvested into 600 ml. plastic containers and the cells are allowed to settle over a period of 10 to minutes. The excess media is then decanted and the remaining sedimentary phase is centrifuged at 2,000 r.p.m. for minutes at 5 to 10 C. The supernate is then discarded. The sediments contain whole viable cells of Mycobacterium phlei. The cells are washed three times by thorough resuspension in 200 to 300 ml. distilled water and centrifugated for 10 to 15 minutes at 2,000 r.p.m.

Example 2 To a freshly harvested cell suspension, containing approximately 135 g. of dry cells, in 2 litres of 0.5 percent of Tween 80 in water in a modified Eppenbach colloid mill is added 1,200 ml. of superfine glass beads of an average dimension of 120 microns. The aperture of the mill is set at 0.03 inch and the suspension is then passed through the mill and recirculated for a period of minutes at a temperature of 0 to 10 C. The ruptured cell suspension is then collected in a flask and the mill is thoroughly washed out with one 150 ml. portion of 0.5% Tween 80. The glass beads are then allowed to settle and the supernatant fluid containing the ruptured cell suspension is decanted. The decanted ruptured cell suspension is kept at approximately 5 C.

Example 3 The ruptured cell suspension, as obtained in Example 2, is centrifuged at 9,200 to 11,000 times gravity for to minutes of 5 to 10 C. At the conclusion of this treatment, the supernatant fluid is discarded and the sediment resuspended with distilled water to the original volume. The reconstituted suspension is now centrifuged at 1,000 to 1,200 gravity at 5 to 10 C. for approximately 30 minutes. The supernatant is decanted and is stained with an acid-fast stain, such as Ziehls Carbol- Fuchsin, to determine whether any intact cells remain. If

there are intact cells remaining, the supernatant is recentrifuged at 1,000-1,200 g. until is entirely cell-free. The isolated cell-free material remaining in the supernatant is then kept frozen at a temperature of 20 to 25 C.

Example 4 The total solids present in the cell-free suspension, obtained as in the previous example, are determined by placing a measured aliquot of cell suspension in a t-a-red vessel and drying it at 90 C. until a constant weight is obtained. To this cell-free suspension, after thawing at room temperature, is added 1 mg. of ribonuclease per 20 mg. of total solids. The treated suspension is then incubated at 37 C. for 60 minutes. At the end of the incubation, the suspension is centrifuged at 9,200-11,000 times gravity at 5 to C. for a period of 30 minutes, or until a separation of phases occurs. At the end of the end of the centrifugation the supernatant is removed by suction and discarded. The sediment, containing cell wall components by Mycobacterium phlei, is resuspended in distilled water, centrifuged at 9,200-11,000 times gravity and the supernatant is again discarded. The sediment is resuspended to its original volume with distilled water and again the total solids present are determined. To this reconstituted suspension is added 2.5 mg. of trypsin per 8.0 mg. of total solids. The resulting suspension is thoroughly mixed and incubated for 60 minutes in a 37 C. water bath. At the end of the incubation, the suspension is treated as described above after the ribonuclease digestion procedure. The resulting sediment is resuspended to half of its original volume with distilled water and the total solids present are determined by placing a measured aliquot in a tared vessel and drying it at 90 C. until a constant weight is obtained. This suspension contains the active reticulo-endothelial stimulant.

Example 5 Mice injected intravenously with 0.4 mg. of the purified cell wall extract of Mycobacterizlm phlei, contained in 0.2 ml. of distilled water, demonstrated a 10 day increase in median survival time after being challenged with a lethal dose, for controls, of Salmonella enteritidis over those mice which did not receive the Mycobacterz'um phlei extract.

Example 6 Mice receiving 0.4 mg. of the purified extract of lllycobacterium phlei intravenously are challenged with a 100% lethal dose of Salmonella enteritidz's eight days later. 80% of the mice survived after the Salmonella infection and they are sacrificed 45 days after being infected. Examination of their livers shows a marked reticulo-endothelial hyperplasia in the Kupifer spaces. This is rather prominent and distinct and the cells form sheets and masses. Hyperplasia of the reticulo-endothelial system elements is pronounced. This indicates a definite stimulation of the reticulo-endothelial system.

Example 7 Mice receiving 0.25 mg. of the purified extract of Mycobacterz'um phlei intravenously are challenged with a 100% lethal dose of Salmonella enteritidis eight days later. Of the nine mice surviving after the Salmonella infection, five are sacrificed 45 days after being infected. Examination of their spleens shows the capsule and trabecular tissue of the spleens to be in normal appearance. Lymphoid follicles are prominent and in some areas appear to coalesce. The most striking feature, however, is the profound reticulo-endothelial hyperplasia in the red pulp. Cords of reticulo-endothelial cells are so closely packed as to almost completely compress the venus-sinusoids. Within these venus-sinusoids there are many plasma cells and plasma blasts and. monocytes. There are also some megakaryocytes present. Within the lymphoid follicles there are some germinal centers composed of hematopoietic reticulo-endothelial cells. In the periphery of the follicles there are mature lymphocytes seen. The arterial trees of the spleens are normal in appearance. The most striking feature, however, is the profound reticulo-endothelial hyperplasia far beyond anything normally seen, indicating a definite reticuloendothelial system stimulation.

Example 8 Sections from the spleens of female mice injected intravenously with 0.25 mg. of the purified extract of Mycobacterium phlei are examined histologically 107 days after treatment. The combined spleen-liver weights of the three animals examined are as follows:

Mg. Spleen weight of mouse No. l 250 Liver weight of mouse No. 1 1600 Spleen weight of mouse No. 2 200 Liver weight of mouse No. 2 1900 Spleen weight of mouse No. 3 232 Liver Weight of mouse No. 3 2450 Microscopic examination shows a thin capsule and normal trabecular pattern which is not as active as in the 45 day specimen. The lymphoid follicles are prominent but the germinal centers are not as active as in the 45 days specimen. In the white pulp there is still a moderate reticulo endothelial hyperplasia but this is much less than in the 45 day specimen and the venous sinusoids which were found to be obliterated in the 45 day specimen are now seen lined by normal littoral epithelium. The number of megakaryocytes is normal.

Sections of the liver show a normal capsule. The parenchymal tissue is normal in appearance. Within the liver there are foci of lymphocytes and some reticuloendothelial cells in the parenchyma and in the portal areas. The foci in the portal areas are an instance of lymphocytosis. An occasional multinucleated giant cell having the appearance of megakaryocytes is present within the liver. The descriptions are applicable to all three livers and spleens submitted.

It appears that after 107 days there is a regression of the reticulo-endotnelial cell hyperplasia to a great degree.

Having described our inventon, what we desire to secure by Letters Patent is:

1. In a process for isolating cell wall components from Mycobacterium phlei which comprises:

(a) rupturing said M. phlei by feeding a viable suspension of said M. plzlei in an aqueous vehicle containing at least one surface active agent and discrete particles of an inert material having a maximum average dimension of about microns through an opening of not more than 0.03 inch formed by two surfaces moving at high speed and in opposing directions with respect to each other;

(12) removing the resulting suspension of said ruptured M. phlei from said opening;

(c) subjecting said ruptured M. phlei to a gravitational force of about 9,200 to 11,000 times gravity to obtain a clear supernatant and a sedimentary phase;

(d) discarding said clear supernatant;

(e) suspending said semidentary phase with water containing at least one surface active agent;

(f) subjecting said suspended sedimentary phase to a second gravitational force of about 1,000 to 1,200 times gravity to obtain a supernatant containing isolated cell wall fractions of said M. phlei and sedi 7 mentary phase containing any intact cells of said 114, phlei;

(g) discarding said sedimentary phase containing in tact cells, and

(h) purifying said supernatant fluid containing isolated cell wall fractions of M. phlei by digesting with ribonuclease at a temperature of 37 to 38 C., separating the ribonuclease digested cell Wall fraction from ribonuclease, digesting said ribonuclease digested cell wall fraction with trypsin at a temperature of 37 to 38 C. and separating the resulting trypsin digested cell wall fraction from trypsin.

2. A composition for parental injection comprising an aqueous suspension of cell wall fractions separated from Mycobacterium phlei obtained in accordance with claim 1.

References Cited in the file of this patent UNITED STATES PATENTS 2,175,090 Parfentjev Oct. 3,

5 2,432,970 Pingert et al. Dec. 16, 2,756,176 Mauser et a1. July 24, 3,001,913 Beers Sept. 26,

OTHER REFERENCES 10 Clifton et al.: Annual Review of Microbiology, vol. 1,

King et al.: Journal of General Microbiology, pp. 315- 324 (September 1948).

Science, vol. 112, pp. 618620 (1950). 5 OConnor et al.: Journal of Bacteria, vol. 73, No. 3,

pp 303-313 (March 1953). 

1. IN A PROCESS FOR ISOLATING WALL COMPONENTS FROM MYCOBACTERIUM PHLEI WHICH COMPRISES: (A) RUPTURING SAID M. PHLEI BY FEEDING A VIABLE SUSPENSION OF SAID M. PHLEI IN AN AQUEOUS VEHICLE CONTAINING AT LEAST ONE SURFACE ACTIVE AGENT AND DISCRETE PARTICLES OF AN INERT MATERIAL HAVING A MAXIMUM AVERAGE DIMENSION OF ABOUT 120 MICRONS THROUGH AN OPENING OF NOT MORE THAN 0.03 INCH FORMED BY TWO SURFACES MOVING AT HIGH SPEED AND IN OPPOSING DIRECTIONS WITH RESPECT TO EACH OTHER; (B) REMOVING THE RESULTING SUSPENSION OF SAID RUPTURED M. PHLEI FROM SAID OPENING; (C) SUBJECTING SAID RUPTURED M. PHELI TO A GRAVITATIONAL FORCE OF ABOUT 9,200 TO 11,000 TIMES GRAVITY TO OBTAIN A CLEAR SUPERNATANT AND A SEDIMENTARY PHASE; (D) DISCARDING SAID CLEAR SUPERNATANT; (E) SUSPENDING SAID SEMIDENTARY PHASE WITH WATER CONTAINING AT LEAST ONE SURFACED ACTIVE AGENT; (F) SUBJECTING SAID SUSPENDED SEDIMENTARY PHASE TO A SECOND GRAVITATIONAL FORCE OF ABOUT 1,000 TO 1,200 TIMES GRAVITY TO OBTAIN A SUPERNATANT CONTAINING ISOLATED CELL WALL FRACTIONS OF SAID M. PHLEI AND SEDIMENTARY PHASE CONTAINING ANY INTACT CELLS OF SAID M. PHLEI; (G) DISCARDING SAID SEDIMENTARY PHASE CONTAINING INTACT CELLS, AND (H) PURIFYING SAID SUPERNATANT FLUID CONTAINING ISOLATED CELL WALL FRACTIONS OF M. PHLEI BY DIGESTING WITH RIBONUCLEASE AT A TEMPERATURE OF 37* TO 38*C., SEPARATING THE RIBONUCLEASE DIGESTED CELL WALL FRACTION FROM RIBONUCLEASE, DIGESTING SAID RIBONUCLEASE DIGESTED CELL WALL FRACTION WITH TRYPSIN AT A TEMPERATURE OF 37* TO 38*C. AND SEPARATING THE RESULTING TRYSPIN DIGESTED CELL WALL FRACTION FROM TRYPSIN. 